The present invention relates generally to variant prothrombins and thrombins capable of activating protein C and having substantially reduced fibrinogen cleavage activity. More specifically, the invention relates to antithrombotic variant prothrombins and thrombins that have substantially reduced procoagulant activity, and to methods of reducing thrombus formation by administering the antithrombotic variant prothrombins or thrombins to an animal or human.
Thrombosis is caused by fibrin and platelet deposits that occlude blood vessels in various organs. The role of thrombosis in morbidity and mortality has been documented in many diseases, including, among others, deep vein thrombosis, pulmonary thrombo-embolism, myocardial infarction, ischemic stroke, anthrax and meningococcal sepsis, and heparin-induced thrombocytopenia. Macrovascular thrombosis can be prevented or successfully treated with anticoagulants, antiplatelet agents, and/or profibrinolytic agents. The antithrombotic therapy for microvascular thrombosis, however, presents a greater medical challenge. Pharmacological use of activated protein C (APC), a naturally circulating anticoagulant enzyme (Gruber et al. Blood 79: 2340-2348 (1992)) has been shown to reduce the mortality of severe sepsis (Bernard et al. New Eng. J. Med. 344: 699-709 (2001)). Clinical use of APC is now medically justifiable. However, manufacturing of injectable dosage forms of natural or recombinant APC for therapeutic use is expensive, especially in view of the large doses, such as, for example, administering 0.024 mg/kg/hour of recombinant human APC for the several days required for effective treatment.
The activation of naturally occurring physiologic systems leading to the production of endogenous therapeutic proteins can be as efficacious and more economical than administering the directly acting agent itself For example, relatively inexpensive plasminogen activators, such as streptokinase, are valuable in the systemic treatment of thrombosis, while the directly acting enzyme, plasmin, is suitable for topical therapy only (Marder et al. Thromb. Haemost. 86: 739-745 (2001)). An affordable alternative modality is needed to make APC-therapy accessible to a broader patient population, including those who suffer from septic disseminated intravascular coagulation due to sepsis.
Low dose wild-type human thrombin (WT) is a relatively safe antithrombotic agent in baboons (Hanson et al. J. Clin. Invest. 92: 2003-2012 (1993)) that is capable of binding to thrombomodulin and generating endogenous APC. However, the low-grade fibrin formation and platelet activation that accompany the infusion of WT have potentially adverse side-effects. WT not complexed with thrombomodulin can cause potentially fatal disseminated intravascular coagulation (Gresele et al. J. Clin. Invest. 101: 667-676 (1998)). Thrombomodulin deficiency or poor microcirculation in a patient pose additional safety risks. The use of guanidinobenzoyl thrombin for protein C activation has addressed several of these problems of WT because acyl-thrombin yields active thrombin by delayed deacylation after binding to endothelial thrombomodulin (McBane et al. Thromb. Haemost. 74: 879-885 (1995)). Acyl-thrombin is effective with a wider safety margin than WT in a pig model of thrombosis. The acylation approach, however, reduces, but does not eliminate, the potentially disastrous consequences of an inadvertent overdose, when simultaneous deacylation of unbound acyl-thrombin would suddenly clot all circulating blood.
Activation of the circulating protein C pool by a suitable snake venom activator has also been shown to produce antithrombotic effects in an arterio-venous (AV) shunt model (Kogan et al. Thromb. Res. 70: 385-393 (1993)). There are several potential advantages to snake enzymes, such as high specificity, long half-life, and stability. Immunogenecity, however, does present a problem if used repeatedly. Also, these enzymes are not readily available. In search for newer, specific, and safe protein C activators, a number of thrombin mutations have been reported to compromise cleavage of fibrinogen more than the activation of protein C (Cantwell et al. J. Biol. Chem. 275:39827-39830 (2000); Wu et al. Proc. Natl. Acad. Sci. U.S.A. 88:6775-6779 (1991); Gibbs et al. Nature 378: 413-416 (1995); Arosie et al. Biochemistry 39: 8095-8101 (2000)).
What is still needed, however, is an antithrombotic thrombin with a substantially reduced procoagulant activity and a compromised platelet activation activity, but having an effective capability to activate protein C.
What is also needed, therefore, is a variant thrombin that is practically devoid of activity towards fibrinogen and the platelet receptor PAR-1, but retains a significant capability to activate protein C in the presence of thrombomodulin.
What is still further needed are methods of administering to a patient variant thrombins capable of activating protein C but not of inducing thrombus formation.
What are also needed are methods to determine the antithrombotic potential and the status of activated protein C in the blood of an animal or human that do not necessitate the use of expensive protein C activators or the use of a protein C activator capable of inducing thrombus formation.
Briefly described, the present invention relates to novel antithrombotic variants of thrombin capable of proteolytically activating protein C, but which are substantially free of fibrinogen cleavage activity. The present invention further relates to variant prothrombins that may be cleaved to yield active thrombin variants. The present invention also relates to methods of inhibiting thrombus formation in an animal or human subject by delivering an antithrombotic variant thrombin of the present invention to the blood of the subject. The present invention relates also to methods that use the novel variant thrombins for determining the level of protein C activation in a blood sample or the thrombogenic potential of a patient.
The present invention provides variant prothrombins and thrombins that have substantially reduced fibrinogen cleavage activity and retain protein C activation activity. Nucleic acid encoding the variant prothrombins or thrombins of the present invention may be inserted into an expression vector and expressed in eukaryotic host cells. The secreted, glycosylated polypeptides may then be cleaved to the active variant thrombins and purified. The variant prothrombins and thrombins of the present invention are useful for administering to a patient as antithrombotic agents lacking a potent thrombus formation capability.
One aspect of the present invention provides variant prothrombins and thrombins that have a tryptophan-alanine substitution at the W215 position of the wild-type thrombin. The single mutant variant thrombin W215A has a substantially reduced fibrinogen cleavage activity and, therefore, a significantly reduced capability of thrombus formation in vivo, or procoagulant activity if contacted with blood in vitro. The variant thrombin W215A retains much of the activity of protein C activation seen with wild-type thrombin, and has an increased relative specificity with respect to PAR-1 cleavage.
Another aspect of the present invention provides a double mutant variant prothrombin and thrombin, WE, that have alanine substitutions at the W215 and E217 positions. The variant thrombin WE of the present invention has a substantially reduced fibrinogen cleavage activity, and significantly reduced PAR-1 cleavage activity (and hence reduced platelet activation activity). The WE variant retains a significant level of protein C activation activity. This variant thrombin, WE (W215A/E217A), is particularly useful for activating protein C, without either significantly cleaving fibrinogen or activating the PAR-1 under in vitro or in vivo conditions. Variant thrombin WE of the present invention has a significantly reduced capability to induce thrombus formation when delivered to the blood of animal or human patient.
The W215A/E217A substitutions of the WE variant thrombin of the present invention reverses the relative specificity of thrombin towards fibrinogen and protein C. The W215A/E217A mutant is substantially inactive toward the substrates fibrinogen and PAR-1, and does not detectably clot fibrinogen or activate platelets in vivo. The WE variant thrombin recovers almost its full activity toward protein C upon binding to thrombomodulin. The WE variant thrombin of the present invention cleaves and activates protein C in the presence of thrombomodulin at a rate only 6-fold slower compared to wild-type thrombin.
The extremely low activity toward prothrombotic substrates, the insignificant rate of inhibition by antithrombin III and the robust rate of hydrolysis of the anticoagulant substrate protein C in the presence of thrombomodulin endow the WE variant thrombin of the present invention with all the required properties of a desired potent antithrombotic thrombin. The mutant is practically inactive toward natural substrates until it binds to thrombomodulin and therefore it can exert its antithrombotic role in the entire blood circulation, particularly in the heart, the brain and the microcirculatory bed.
The variant prothrombins and thrombins of the present invention are useful antithrombotic, agents suitable for administering to a patient to inhibit thrombus formation by the activation of protein C. The present invention contemplates, that the prothrombins may be administered to an animal or human to be cleaved in vivo to deliver the corresponding variant thrombin to the blood.
Analogous to the endogenous fibrinolytic pathway, the anticoagulant capacity of a pharmacologically inducible endogenous protein C pathway surpasses the level that is needed and safe for therapeutic use. The variant thrombin WE of the present invention is useful for inducing the endogenous APC by the administration of low doses of the variant thrombin WE. This induction can be more efficient than using high-doses of administered exogenous APC, on a dosage-weight basis.
It is contemplated that the variant thrombins of the present invention are useful for determining the protein C activity in an individual. An especially useful variant thrombin is the double mutant WE that is both easier and cheaper to produce than alternative protein C activators such as snake venom. The protein C activity of a blood sample can be determined by using a variant thrombin of the present invention as an activator. One stage and multiple stage tests are contemplated by the present invention. A citrated venous, arterial or capillary blood sample may be added to a variant thrombin of the present invention. Contact between the protein C in the blood sample and the variant thrombin leads to the generation of APC. The resultant APC activity of the solution phase admixture may then measured by standard procedures.
Another assay method contemplated by the present invention is to add the blood sample to the variant thrombin protein C activator, a thrombomodulin analog, and calcium ions, with or without an APC chromogenic substrate. The rate of coagulation or cleavage of the chromogenic substrate will correspond to the level of protein C activity in the sample.
The present invention further contemplates an assay to determine the endogenous antithrombotic potential of the protein C system of an individual following the pharmacological induction of circulating APC activity in the blood. An increase in blood APC levels will follow administration of an effective amount of a protein C activator variant thrombin of the present invention. This global test will reflect the combined function of most components of the protein C system, notably protein C and thrombomodulin, but not of protein S. The subject will receive an effective dose of a protein C activator variant thrombin tol induce a temporary increase in the concentration of circulating APC that can be measured in one or more blood samples taken from the subject. This test, especially when combined with a protein C activity test, permits an assessment of the antithrombotic potential of the protein C pathway of an individual.
The present invention also contemplates a method of preparing activated protein C free of procoagulant and platelet activating activity. A protein C sample such as, for example, a protein C isolated from a biological fluid, or a protein C produced by recombinant DNA technology, can be converted into APC by contact with the protein C activator variant thrombin WE. The process can be terminated by separation of the activator variant thrombin from the substrate protein C, or by quenching the activity of the variant thrombin with an inhibitor. The activation process is particularly useful for manufacture of improved APC preparations from protein C for therapeutic purposes. The use of a variant thrombin, such as WE of the present invention, as the protein C activator ensures that the activated protein C preparation will not induce coagulant activity if administered to a patient.